Establishment of a Protoplast Transformation Method in the Oleaginous Yeast <i>Rhodotorula toruloides</i>

Rhodotorula toruloides is an oleaginous yeast with great potential for chemical and biofuel production, due to its ability to utilize lignocellulosic biomass and to produce high levels of carotenoids and storage lipids. However, its broader application in biotechnology has been limited by the lack of efficient genetic transformation methods. Although protoplast transformation is wildly used in fungal systems, it has remained largely unexplored in R. toruloides. In this study, we established a protoplast transformation protocol using linear DNA fragments and R. toruloides strain BOT-A2, a recently isolated strain with high lipid-producing potential. We first confirmed that BOT-A2 is a MAT A2 haploid strain. We then produced a β-1,3-glucomannanase (Man5C) that effectively digests the R. toruloides cell wall. Key parameters affecting transformation efficiency were systematically optimized, including the Man5C digestion conditions, antibiotic selection pressure, cell growth phase, protoplast yield and viability, PEG formulation, calcium ion concentration, and regeneration conditions. Using the optimized protocol, we successfully transformed BOT-A2 with three heterologous resistance cassettes (hygromycin R, bleomycin, and G418) yielding 190, 226, and 244 transformants per µg of DNA, respectively. This method provides a platform for genetic manipulation and is expected to facilitate both fundamental research and metabolic engineering in R. toruloides BOT-A2.